There is an urgent need for the early detection of a deliberate release of harmful, toxic materials in the air originating from acts of tenor, offensive military action or accidents. Those materials can be of chemical or biological nature. Those materials can be present in the form of aerosol, solid particles, and vapor, or a combination of aerosol, particles and vapor.
In order to minimize the harmful effects of those materials, an early, near real-time detection is essential at low concentrations allowing the early deployment of counter measures and evacuation prior to causing damage to humans or loss of human life. However, presently used technologies and devices have limitations in effectively achieving this early detection of such harmful chemicals or biological threats. Enzyme-based biosensors are most suitable for detection of toxins such as, for example, but not limited to, pesticides, acids, chemical warfare agents, and toxic industrial chemicals because enzymes can be selectively inhibited by a particular class of chemicals. Enzymes are highly specific biocatalysts which are typically not affected by other chemicals present and therefore provide a high resistance to interferences by other chemicals in the environment of question. Enzyme-based biosensors are also used in detecting target chemicals that act as substrates for enzymes. In most biosensors, the sensing enzymes are incorporated in devices such as electrodes, transducers, fiber optics, hydrogels, polymer sponges, or crystals, and the target chemical must be physically contacted with the device so as to enable its interaction with the sensing enzyme.
In general, six classes or types of enzymes (as classified by the type of reaction that is catalyzed) are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases. Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
Enzymes have been known since the early 1960's to be useful tools for detecting the presence of chemical species. Rogers, K. R., Biosensors Bioelectronics, 10, 533 (1995). Generally all enzymatic biosensors function by one of two methods. The enzyme either converts an undetectable compound of interest into another or series of compounds which can be detected with a chemical-based sensor or the enzyme is inhibited by the presence of the compound of interest and the enzyme inhibition is linked to a measurable quantity.
As described above, many biosensors employ the principle of enzyme inhibition by certain class of chemicals. For example, organophosphates inhibit cholinesterases, cyanides inhibit peroxidases, and heavy metals inhibit ureases, and so on. These phenomena cause drastic changes in the chemical and/or physical state of the system and these changes are sensed by the read-out mechanism employed in various biosensors.
Similarly, many enzyme-based sensors detect the target analyte by utilizing the analyte itself as a substrate e.g. glucose, urea, creatinine etc. This phenomenon also causes physical/chemical changes in the sensory system of the biosensor used and enables detection of the target analyte. In the following paragraphs, we briefly review traditional known background enzyme based biosensors for a variety of target analytes, which are based either on enzyme inhibition or on enzyme catalysis.
Acetylcholinesterase Based Organophosphate Sensors:
Depending on the potency, phosphoesters of organic alcohols are used either as insecticides, pesticides or as nerve agents in chemical warfare. In both the applications, enzyme acetyl cholinesterase (AChE) in exposed organisms is irreversibly inhibited due to phosphorylation of serine hydroxyl group in the active site of the enzyme. Detection of trace amounts of organophosphates in agriculture and in civilian and military environment is an important area of research. Numerous amperometric and potentiometric sensors comprising AChE have been reported so far (N. Jaffrezic-Renault, Sensors 1, 60-74 (2001)). In these sensors AChE is immobilized on the surface of transducers through different techniques. In amperometric sensors the current generated by oxidation of thiocholine (which is generated by enzymatic hydrolysis of substrate butyrylthiocholine) is measured. Also, a bi-enzymatic system of AChE and choline oxidase has been used to detect organophosphates, wherein, hydrogen peroxide generated by oxidation of choline (which is generated by AChE catalyzed hydrolysis of acetylcholine) is detected. In case of potentiometric sensors such as ion selective electrodes (ISE) or ion sensitive field effect transistor (ISFET), organophosphates are detected by monitoring the change in the pH due to the acid generated from enzymatic hydrolysis of acetylcholine.
Recently, a photonic crystal-AChE based sensor for organophosphates has been reported. This sensor uses AChE immobilized in a polymerized crystalline colloidal array. When trace amounts of organophosphates inhibit AChE, the polymer swells and changes the lattice spacing in crystals causing red-shift in the wavelength of diffracted light (J. P. Walker, S. A. Asher, Anal. Chem. 77, 1596-1600 (2005)).
U.S. Pat. No. 7,008,524 describes the sensor and the method to detect chemical agents using metal interdigitized electrodes coated with polymer film containing AChE. When organophosphates react with AChE in the sensor, chemical and/or morphological changes occur in the polymer film and this modulates the electric current flowing through the electrode. U.S. Pat. No. 6,821,738 describes optical sensor based on reversible complex of AChE and porphyrins or phthalocyanines. When organophosphates react with AChE, they displace the fluorescent porphyrins from the active site of AChE. This causes changes in the absorption and/or fluorescence spectra of porphyrins that are detectable by spectrophotometers. U.S. Pat. No. 6,541,230 describes polyurethane sponges containing a covalently immobilized AChE, butyrylcholinesterase, organophosphorous hydrolase, and the indicator useful in “verified decontamination” of chemical warfare agents. U.S. Pat. No. 6,750,033 describes polyurethane polymer containing AChE (which is inhibited by organophosphates) and a second base-producing enzyme urease (which is not inhibited by organophosphates). In the absence of organophosphates, the polymer soaked in substrates solution has neutral pH as both the enzymes in the polymer are producing acid and base at controlled rates. When organophosphates are swiped onto the sensor, AChE in the polymer is inhibited and pH of the medium is increased. This is visualized by pH sensitive indicator dye incorporated in the polymer.
Organophosphorous Hydrolase Based Organophosphate Sensors:
Since AChE is irreversibly inhibited by organophosphates the sensors based on AChE are made for single use application. For reusable applications, researchers have developed sensors using organophosphorous hydrolases (OPH) which catalytically hydrolyze organophosphates as their substrates. Mulchandani et al have reviewed the present state of the art in OPH based biosensors which can be broadly categorized into potentiometric, optical and amperometric sensors (A. Mulchandani, W. Chen, P. Mulchandani, J. Wang, K. R. Rogers, Biosensors and Bioelectronics 16, 225-230 (2001)). Potentiometric sensor for organophosphates has been reported by immobilizing a layer of OPH crosslinked with bovine serum albumin and glutaraldehyde on to a pH electrode. The electrode measures change in the pH when it is in contact with the solution containing organophosphates (P. Mulchandani, A. Mulchandani, I. Kaneva, W. Chen, Biosensors and Bioelectronics 14, 77-85 (1999)).
Two different optical sensors containing OPH have been developed. In the first sensor, fluorescein isothiocyanate (FITC) labeled OPH was adsorbed on poly(methyl methacrylate) beads and the sensor beads were contacted with the analyte in a microbead fluorescence analyzer. The presence of organophosphates was detected by monitoring decrease in the fluorescence of FITC label on inhibited AChE. In the second optical sensor, a fiber optic set up was built with desired cut off wavelength of 348 or 400 nm to detect hydrolysis products of organophosphates such as coumaphos or p-nitrophenol, respectively. OPH was immobilized on a nylon membrane and attached to the optical fiber in the set up (A. Mulchandani, S. Pan, W. Chen, Biotechnol. Prog. 15, 130-134 (1999)).
OPH based amperometric sensor for organophosphates has been developed in the form of a screen-printed thick film carbon electrode. The electrode was coated with Nafion membrane containing OPH. p-nitrophenolate anion released by enzymatic hydrolysis of certain organophosphates was oxidized at the anode and the generated current was measured using a potentiostat (A. Mulchandani, P. Mulchandani, W. Chen, J. Wang, L. Chen, Anal. Chem. 71, 2246-2249 (1999)). In a modification of this technique, a remote OPH-based amperometric biosensor was also developed (J. Wang, L. Chen, A. Mulchandani, P. Mulchandani, W. Chen, Electroanalysis 11, 866-869 (1999)).
All these sensors have exhibited very low detection limits ranging between 0.5 to 50 μM concentrations of organophosphates. However, the operating mechanism of these sensors requires that each time the sample must be applied to the electrode or polymer in order to detect the presence of organophosphates. Thus, none of these background sensors are particularly conducive for fully automatic detection in the air. Biosensors comprising enzyme-electrodes and based on enzyme-inhibition have been also developed for drugs, cyanide, heavy metals, and chemicals.
Peroxidase Based Cyanide Sensors:
Horseradish peroxidase (HRP) is reversibly inhibited by cyanide ions. Therefore, HRP based biosensors have been constructed by many researchers to monitor the cyanide traces in water. For example, HRP immobilization on surface of ISFET has been reported (V. Volotovsky, N. Kim, Biosensors and Bioelectronics 13, 1029-1033 (1998). The sensor was constructed by coating the electrode with HRP immobilized in poly(4-vinyl pyridine-co-styrene). The sensor was able to detect 0.6 μM potassium cyanide and was able to be reused after washing. Similarly, HRP based amperometric sensor has been reported by immobilizing the enzyme and an osmium redox polymer ([Os(bipyridyl)2(poly(vinyl pyridine)10Cl]Cl) on to an electrode. Upon addition of substrate hydrogen peroxide, a biocatalytic reduction generated the current. This current was inhibited by the analyte cyanide to cause change from 150 mV to 0 mV. Cyanide detection ranged between 4 μM to 40 μM (T-M. Park, E. I. Iwuoha, M. R. Smyth, Electroanalysis 9, 1120-1123 (1997)). A cyanide sensor electrode based on cytochrome oxidase has been also reported (A. Amine, M. Alafendy, J-M. Kauffmann, M. N. Pekli, Anal. Chem. 67, 2822-2827 (1995)). Here also, these sensors are limited to detect cyanide in aqueous samples and are not conducive to determine the presence of cyanide on surfaces.
Urease Based Heavy Metal Ion Sensors:
Urease is inhibited by toxic heavy metal ions such as mercury, lead, and cadmium. Thus, urease based sensors have been constructed to detect trace amounts of heavy metal ions in drinking water and industrial effluents. For example, a conductometric urease biosensor has been reported for detection of Hg+2, Cu+2, Cd+2, and Pb+2 ions (S-M. Lee, W-Y. Lee, Bull. Korean Chem. Soc. 23, 1169-1172 (2002)). The sensor was constructed by immobilizing the enzyme-silica sol-gel as a thick film on screen printed interdigitated array electrode. Inhibition of urease by heavy metal ions was measured from the difference in the admittance response for 1 mM urea before and after the interaction with metal ions. Also, urease based optical biosensor for heavy metals have been constructed by immobilizing the enzyme on aminopropyl glass. Heavy metals were detected by monitoring changes in pH resulting from urease catalyzed hydrolysis of urea before and after the incubation with metal ions (R. T. Andres, R. Narayanaswamy, The Analyst, 120, 1549-1554 (1995)).
Enzyme Based Toxic Chemical Sensors:
Amperometric sensors for detection of thiols, carbamates, thiourea, and benzoic acid have been reported by using tyrosinase and peroxidase electrodes (J. Wang, E. Dempsey, A. Eremenko, Anal. Chim. Acta 279, 203-208 (1993)). Aqueous solutions of enzyme and crosslinking polymer were applied to an electrode to form the enzyme-containing film layer around the electrode. Detection of chemicals was performed by measuring the current generated upon addition of 0.2 mM phenol before and after the incubation with the chemical.
Enzyme Based Sensors for Analytes Used as Substrates:
Urease catalytically hydrolyzes urea into ammonia, carbon dioxide and water. Urea can be present as adulterant in milk. Also, urea can be present in river water and in industrial effluents. Therefore, urease based biosensors have been developed to detect urea in various aqueous samples. For example, potentiometric urea sensor has been developed by coating the surface of a microelectrode with crosslinking mixture of urease, polyethyleneimine, and glutaraldehyde. The sensor exhibited short response time (15-30 seconds) and linear detection range of 1-100 mM urea (Lakard, B., Herlem, G., Lakard, S., Antoniou, A., Fahys, B., Biosensors and Bioelectronics 19, 1641-1647 (2004)).
Similarly, amperometric urea biosensor has been developed by immobilizing urease-containing conducting polymer film of poly((N-3-aminopropyl pyrrole-co-pyrrole) onto an electrode (Bisht, R. V., Takashima, W., Kaneto, K., Biomaterials 26, 3683-3690 (2005)). The electrode measured the redox current generated by pH sensitive redox compound hematein. The electrode gave linear response in the range of 0.16-5.0 mM urea in aqueous medium.
For diabetic patient populations, glucose is an important analyte detected by glucose oxidase based biosensors that oxidize the substrate glucose into gluconic acid. A variety of blood glucose sensors are available in the market that are based on glucose oxidase. Apart from this, there is continuously ongoing research in the field of glucose sensors to improve sensor operations and patient comfort levels. For example, glucose sensing contact lens has been reported to monitor glucose levels in tears (Badugu, R., Lakowicz, J. R., Geddes, C. D. Journal of Fluorescence 13, 371-373 (2003)). The contact lens uses boronic acid containing flourophore which reacts with vicinal diols in glucose and changes its fluorescence to detect 0.05-1.0 mM glucose in tears, which can be tracked to 5-10 fold higher glucose level in the blood.
Creatinine is yet another important health-biomarker which can be detected using enzyme based sensors. For example, potentiometric biosensor for creatinine has been reported by using electrode modified with creatinine deiminase, the enzyme that degrades creatinine to produce ammonia (Shih, Y., Huang, H., Anal. Chim. Acta 292, 143-150 (1999)). Interestingly, creatinine amidohydrolase, the enzyme that converts creatinine into creatine has been also used to develop an amperometric biosensor (Berberich, J. A., Chan, A., Boden, M., Russell, A. J., Acta Biomaterialia 1, 193-199 (2005).
The development of monitoring devices for sampling and for chemical identification and detection has also been previously put to practice. Much of the art related to device development focuses on equipment for use in laboratories as automated samplers or fluid handling equipment. U.S. Pat. Nos. 4,224,033 and 4,338,280 each describe fluid handling devices that facilitate hands-free processing of individual samples in a preparatory fashion for later analysis and evaluation. Similarly, U.S. Pat. No. 4,066,412 discloses a device that can carry disposable reagents to aid in monitoring the physical properties of a reaction mixture by passing through a fixed path length.
Other background art describes devices that employ specialized components to facilitate the use of particular sensing chemistries and protocols for fluid analysis. U.S. Pat. No. 4,826,759 describes a fluid sampling device that carries two absorbent layers that are used to bring fluid components into the device and transfer such elements to a second layer for chemical analysis. U.S. Pat. Nos. 4,726,929 and 4,958,295, describe modular devices that handle and analyze fluids in unique ways including disposable collection modules and internal vacuum drives, respectively.
U.S. Pat. No. 4,525,704 describes the use of cholinesterase and electrical currents in detecting toxic gases. Other patents describe devices that can be used to detect the presence of enzyme substrates within a specified sample. U.S. Pat. No. 5,223,224 describes an arrangement for flow injection analysis which sample gases are kept isolated from the environment within the device. U.S. Pat. Nos. 5,504,006 and 5,994,091 both describe sensor devices to sample gas and liquid streams, respectively, for enzyme substrates by linking enzyme activity brought on by the presence of substrate to a colorimetric signal. U.S. Pat. No. 7,422,892 B2 describes another device that employs an enzyme and substrate pair to continuously monitor an incoming sample for the presence of an enzyme inhibitor. This sensor includes an immobilized enzyme that is selected to be inhibited by the analyte. This device also includes a mechanism to continuously or semi-continuously deliver a substrate compound to the immobilized enzyme.
All of the background art mentioned above for enzyme based sensors can detect the analyte of interest when it is present in the sample solution applied to the sensor. But none of these background art sensors have the ability to detect the analyte present in the air at near real-time and at very low concentrations, or to detect analyte present in the form of vapor, aerosol and solid particles. Therefore there is a great need in the commercial marketplace to provide a device and a method to monitor air in near real time for the presence of particles, aerosols and/or vapor, and especially for a device that employs an enzyme or enzymes to detect the presence of an enzyme inhibitor within the environment without the active involvement of the user.
U.S. Patent Publication. No 2006/0238757 A1 (“Silcott US Pub. '757) describes a device for detecting, classifying and identifying airborne biological and non-biological particles on an individual basis in near real-time, based on a single particle's intrinsic optical properties. However, this device has several shortcomings. Silcott US Pub. '757 describes a method requiring a reaction environment between the sampled airborne particles and optical reporters pre-reacted with selected markers wherein the reacted optical reporters are adsorbed onto the surface of an aerosol particle. Further, the method disclosed in Silcott US Pub. '757 does not concern itself with providing a cyclic process of continued detection. Silcott US Pub. '757 teaches at great lengths processes for establishing attachment via adsorption of the pre-reacted optical reporter to the surface of the sampled airborne particles. Specifically, Silcott US Pub. '757 teaches a reaction environment between the sampled airborne particles and the pre-reacted optical reporters that is created by either growing a liquid layer onto the airborne sample's particle's surface using evaporation/condensation, molecular sublimation or aerosol coagulation techniques, or by collecting airborne particles and introducing the collected particles to a liquid thin-film. Silcott US Pub. '757 teaches techniques for controlling the thickness and chemical composition of the liquid layer so that the optical reporter, solvent, and other required reagents are successfully adsorbed onto the surface of the aerosol airborne particle. Reacted and non-reacted airborne particles are introduced one at a time in the optical sensor of Silcott US Pub '757 for detection.
Therefore, a need exists for improved methods and devices for detecting, classifying, and identifying airborne biological and non-biological particulates, and discriminating specific biological and non-biological particulates from commonly encountered background particulates.